1. Field of the Invention
This invention relates to apparatuses and methods for use in the treatment of biological and non-biological vesicles which utilize electric field pulses to prealign vesicles, to introduce pores in and through the membranes of vesicles for the purpose of loading or unloading materials into or from the vesicles or for the purpose of fusing of two or more vesicular structures together and, in particular, to apparatuses and methods which utilize homogeneous and uniform electric fields to treat vesicles.
2. Description of the Prior Art
It is known that stable pores may be created in cell membranes, or in other vesicles, by the application of electric field pulses across a liquid cell suspension containing the vesicles. This is referred to as "poration" or "electroporation".
In such poration processes, cells are suspended in any liquid media, electrolyte, non-electrolyte, or mixtures of electrolytes and non-electrolytes and then subjected to an electric field pulse. Pulse lengths, that is the time that the electric field has been applied to such cell suspensions, have varied in length, for example anywhere from about 10 nanoseconds to about 100 milliseconds. The strength of the electric fields applied to suspensions during such poration processes has varied from between about 100 V/cm to about 30 KV/cm. Sale and Hamilton, "Effects of High Electric Fields on Micro-Organisms III. Lysis of Erthyrocytes and protoplasts", Biochimica Et Biophysica Acta, 163 (1968) 37-43. In each instance, in order to create a pore in a cell's outer membrane, the electric field has been applied for such a length of time and at such a voltage that a set potential (a transmembrane potential between about 0.5 volt and about 2.5 volts) has been created across the membrane for a length of time adequate to create a pore in the membrane, as is well known in the art. The electric field created across a 100 angstrom the membrane of a vesicle at 1 volt transmembrane potential is about 1 megavolt per centimeter.
There are four phenomena taught by the prior art which are relevant to the present invention. The first is the phenomenon of dielectric breakdown, that is, the ability of a high electric field to create a small hole or pore in a thin membrane. Once a vesicle is porated it can be loaded or unloaded.
Zimmerman et al, in U.S. Pat. No. 4,081,340 discloses that the "permeability" of cell membranes of cells in an electrically conductive solution can be increased by pumping them through an aperture separating two discrete volumes of electrolyte solution and two electrodes, wherein the electrodes apply an electric field to the cells as they traverse the aperture. As will be set forth in greater detail below, the present invention does not "increase the permeability" of cell membranes, but rather puts actual holes or pores in cell membranes as taught by Kinosita and Tsong, "Formation and Resealing of Pores of Controlled Sizes in Human Erythrocyte Membrane", Nature, Vol. 268, pg. 438-441, Aug. 4, 1977. In "Hemolysis of Human Erythrocytes by a Transient Electric Field", Proc. Natl. Acad. Sci USA, Vol. 74, No. 5, pg. 1923-1927, May 1977, Kinosita and Tsong describe the use of a homogeneous, uniform electric field to create pores in the erythrocyte membrane. And, Kinosita and Tsong in Bibliotheca "Use of Voltage Pulses for the Pore Opening and Drug Loading", (Karger, Basel 1985) Haematologica, No. 51, pp. 108-114 further describe pulse poration of red blood cells for drug loading purposes using 1 to 5 KV/cm uniform electric field pulses of 1 to 200 microsecond duration. Whereas Kinosita and Tsong outline apparatus for processing microliter quantities of red blood cells, no provision is made or suggestion of how one would process quantities of cells in the many milliliter range. Their instrumentation is limited to small volumes due to the impedance (200 ohms) of the pulse instrument.
In Zimmerman U.S. Pat. No. 4,289,756 porated cells, especially erythrocytes, are loaded with therapeutic drugs. In being subjected to such loading, the general morphology of the erythrocytes is greatly disturbed so that the erythrocytes are preferentially accumulated in the spleen and liver. Zimmerman et al., "The Effect of Encapsulation in Red Blood Cells on the Distribution of Methotrexate in Mice", J. Clin. Chem. Clin. Biochem, Vol. 16, 1978, pp. 135-144.
The second phenomenon is a dielectrophoretic bunching effect as taught by F. Pohl in his book, Dielectrophoresis, Cambridge Press 1978, Chapter 4, pp. 39, which describes the mutual self attraction produced by the placement of vesicles in a uniform electric field. Stolley, German Democratic Republic Patent No. 0433-6461, 1984, uses the Pohl technique in a system for collecting suspended particles, especially biological cells for the purposes of hybrid cell technology. Stolley's system uses a chamber having parallel electrodes to generate a homogeneous high frequency electric field to produce dielectrophetic bunching. This phenomenon is different from the dielectrophoretic force achieved by attracting vesicles to an electrode through the use of non-homogeneous field generation.
The third phenomenon is that of vesicle fusion, which is taught by E. Neumann et al. in the paper "Cell Fusion Induced by High Electric Impulses Applied to Dictyostelium", Naturwissenchaften 67, S. 414 (1980), and also by U. Zimmerman et al. in his paper "Fusion of Avena Sativa Mesophyll Protoplasts by Electric Breakdown", Biochimica et Biophysica Acta, 641 (1981, p. 160-165). These papers describe the tendency for membranes of biological vesicles, each of which have had holes or pores formed by dielectric breakdown, to couple together at their mutual dielectric breakdown sites when two such vesicles are in close proximity. The papers also report the natural resultant joining, or fusing of the two cells into a single cell package.
Another prior art process for fusing cells has been presented in an article appearing in the J. Membrane Biol. 67, 165-182 (1982), Electric Field-Induced Cell-to-Cell Fusion by Zimmermann and Vienken. ln this process, disturbances in the membrane structure between neighboring cells are produced by using a penetrating electrical impulse which leads to a cytoplasmic continuum in the membrane contact zone between the two cells and to the creation of a lipid jointure between the membranes of neighboring cells. Due to surface energy reasons, the structure formed as the cells are connected to each other by lipid material is rounded off after a jointure is formed. In order to execute this procedure, a chamber was used which had a volume for containing the cell suspension which is contained by non-conductive walls. At least two metallic electrodes protrude into this area to form a space in which the cells are exposed to an electrical field created by the electrodes. These electrodes are shaped to purposefully generate non-uniform fields. At the same time, the electrodes are connected to a device producing electrical voltage impulses in order to achieve electrical permeation.
In U.S. Pat. Nos. 4,476,004 and 4,441,972 Herbert Pohl describes methods and apparatus for causing electrofusion of biological particles. The descriptions of the process and apparatus in these patents suggest that in order to achieve cell fusion it is required, and of primary importance, to place the suspensions of polarizable cells or vesicles into chambers in which non-homogeneous or non-uniform electric fields are generated.
The fourth phenomenon is the tendency of aspherial cells to line up along one of their axes in the presence of high frequency fields as taught by Teixeira-Pinto et al., "The Behavior of Unicellular Organisms in an Electromagnetic Field", Experimental Cell Research 20, 548-564 (1960).
A process of aligning cells in an electrical field was made known in the Biochimica et Biophysica Acta, 694 (1982), 227-277 "Electric Field-Mediated Fusion and Related Electrical Phenomena, U. Zimmerman". In this process membrane contact between at least two cells is brought about by using an alternating and generally weak inhomogeneous electric field. Dipoles are produced by the electrical fields due to polarization processes in the cell. Such polarized cells display a mutual attraction as the cells draw closer together when migrating in the electrical field, in a form of so-called dielectrophorosis.
In most of the references discussed above, the electric fields utilized are non-uniform using electrode configurations of pin-pin, pin-plate, and wire-wire. See Pohl, Dielectrophesis, supra at 356-359. The use of non-homogeneous, non-uniform electric fields for vesicle treatment represents the majority view that vesicles more properly are treated by the actual non-uniformity of the fields. Yields, such as for poration, unfortunately are low. A minority view, expressed in the Kinosita and Stolley references, supra, utilize homogeneous, uniform electric fields using parallel plate electrodes for the limited treatment of vesicles . Stolley performs dielectrophoretic bunching and Kinosita performs poration and loading of vesicles.
The apparatuses and methods of the present invention utilize homogeneous, uniform electric field generation for treatment of biological and non-biological vesicles including prealignment of vesicles, poration of vesicles, loading of vesicles (and unloading) and fusion of vesicles. The present invention improves upon the teachings of Stolley and Kinosita by providing: (a) rotational prealignment to increase the uniformity of poration of vesicles and the loading of vesicles over a whole sample providing high yield rates, (b) collection of vesicles in a uniform, homogeneous electric field in conjunction with a fusion pulse also generated uniformly and homogeneously, (c) processing of much greater volumes of vesicles and in much lower conductivity suspensions by switching large power signals at very fast switching times, and (d) an apparatus for magnetically producing uniform, homogeneous electric fields in an electrodeless chamber. None of these features of the present invention are found in any of the prior art references.